Biochemical Characterisation and Structure Determination of a Novel Cold-Active Proline Iminopeptidase from the Psychrophilic Yeast, Glaciozyma antarctica PI12
Microbial proteases constitute one of the most important groups of industrially relevant enzymes. Proline iminopeptidases (PIPs) that specifically release amino-terminal proline from peptides are of major interest for applications in food biotechnology. Proline iminopeptidase has been extensively ch...
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ftmdpi:oai:mdpi.com:/2073-4344/12/7/722/ 2023-08-20T04:02:26+02:00 Biochemical Characterisation and Structure Determination of a Novel Cold-Active Proline Iminopeptidase from the Psychrophilic Yeast, Glaciozyma antarctica PI12 Shazilah Kamaruddin Rohaiza Ahmad Redzuan Nurulermila Minor Wan Mohd Khairulikhsan Wan Seman Mahzan Md Tab Nardiah Rizwana Jaafar Nazahiyah Ahmad Rodzli Mohd Anuar Jonet Izwan Bharudin Nur Athirah Yusof Doris Quay Huai Xia Nor Muhammad Mahadi Abdul Munir Abdul Murad Farah Diba Abu Bakar 2022-06-30 application/pdf https://doi.org/10.3390/catal12070722 EN eng Multidisciplinary Digital Publishing Institute Biocatalysis https://dx.doi.org/10.3390/catal12070722 https://creativecommons.org/licenses/by/4.0/ Catalysts; Volume 12; Issue 7; Pages: 722 microbial proteases proline specific protease cold-active enzyme gluten hydrolysis protein structure Text 2022 ftmdpi https://doi.org/10.3390/catal12070722 2023-08-01T05:33:44Z Microbial proteases constitute one of the most important groups of industrially relevant enzymes. Proline iminopeptidases (PIPs) that specifically release amino-terminal proline from peptides are of major interest for applications in food biotechnology. Proline iminopeptidase has been extensively characterised in bacteria and filamentous fungi. However, no similar reports exist for yeasts. In this study, a protease gene from Glaciozyma antarctica designated as GaPIP was cloned and overexpressed in Escherichia coli. Sequence analyses of the gene revealed a 960 bp open reading frame encoding a 319 amino acid protein (35,406 Da). The purified recombinant GaPIP showed a specific activity of 3561 Umg−1 towards L-proline-p-nitroanilide, confirming its identity as a proline iminopeptidase. GaPIP is a cold-active enzyme with an optimum activity of 30 °C at pH 7.0. The enzyme is stable between pH 7.0 and 8.0 and able to retain its activity at 10–30 °C. Although GaPIP is a serine protease, only 25% inhibition by the serine protease inhibitor, phenylmethanesulfonylfluoride (PMSF) was recorded. This enzyme is strongly inhibited by the presence of EDTA, suggesting that it is a metalloenzyme. The dimeric structure of GaPIP was determined at a resolution of 2.4 Å. To date, GaPIP is the first characterised PIP from yeasts and the structure of GaPIP is the first structure for PIP from eukaryotes. Text Antarc* Antarctica MDPI Open Access Publishing Catalysts 12 7 722 |
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English |
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microbial proteases proline specific protease cold-active enzyme gluten hydrolysis protein structure |
spellingShingle |
microbial proteases proline specific protease cold-active enzyme gluten hydrolysis protein structure Shazilah Kamaruddin Rohaiza Ahmad Redzuan Nurulermila Minor Wan Mohd Khairulikhsan Wan Seman Mahzan Md Tab Nardiah Rizwana Jaafar Nazahiyah Ahmad Rodzli Mohd Anuar Jonet Izwan Bharudin Nur Athirah Yusof Doris Quay Huai Xia Nor Muhammad Mahadi Abdul Munir Abdul Murad Farah Diba Abu Bakar Biochemical Characterisation and Structure Determination of a Novel Cold-Active Proline Iminopeptidase from the Psychrophilic Yeast, Glaciozyma antarctica PI12 |
topic_facet |
microbial proteases proline specific protease cold-active enzyme gluten hydrolysis protein structure |
description |
Microbial proteases constitute one of the most important groups of industrially relevant enzymes. Proline iminopeptidases (PIPs) that specifically release amino-terminal proline from peptides are of major interest for applications in food biotechnology. Proline iminopeptidase has been extensively characterised in bacteria and filamentous fungi. However, no similar reports exist for yeasts. In this study, a protease gene from Glaciozyma antarctica designated as GaPIP was cloned and overexpressed in Escherichia coli. Sequence analyses of the gene revealed a 960 bp open reading frame encoding a 319 amino acid protein (35,406 Da). The purified recombinant GaPIP showed a specific activity of 3561 Umg−1 towards L-proline-p-nitroanilide, confirming its identity as a proline iminopeptidase. GaPIP is a cold-active enzyme with an optimum activity of 30 °C at pH 7.0. The enzyme is stable between pH 7.0 and 8.0 and able to retain its activity at 10–30 °C. Although GaPIP is a serine protease, only 25% inhibition by the serine protease inhibitor, phenylmethanesulfonylfluoride (PMSF) was recorded. This enzyme is strongly inhibited by the presence of EDTA, suggesting that it is a metalloenzyme. The dimeric structure of GaPIP was determined at a resolution of 2.4 Å. To date, GaPIP is the first characterised PIP from yeasts and the structure of GaPIP is the first structure for PIP from eukaryotes. |
format |
Text |
author |
Shazilah Kamaruddin Rohaiza Ahmad Redzuan Nurulermila Minor Wan Mohd Khairulikhsan Wan Seman Mahzan Md Tab Nardiah Rizwana Jaafar Nazahiyah Ahmad Rodzli Mohd Anuar Jonet Izwan Bharudin Nur Athirah Yusof Doris Quay Huai Xia Nor Muhammad Mahadi Abdul Munir Abdul Murad Farah Diba Abu Bakar |
author_facet |
Shazilah Kamaruddin Rohaiza Ahmad Redzuan Nurulermila Minor Wan Mohd Khairulikhsan Wan Seman Mahzan Md Tab Nardiah Rizwana Jaafar Nazahiyah Ahmad Rodzli Mohd Anuar Jonet Izwan Bharudin Nur Athirah Yusof Doris Quay Huai Xia Nor Muhammad Mahadi Abdul Munir Abdul Murad Farah Diba Abu Bakar |
author_sort |
Shazilah Kamaruddin |
title |
Biochemical Characterisation and Structure Determination of a Novel Cold-Active Proline Iminopeptidase from the Psychrophilic Yeast, Glaciozyma antarctica PI12 |
title_short |
Biochemical Characterisation and Structure Determination of a Novel Cold-Active Proline Iminopeptidase from the Psychrophilic Yeast, Glaciozyma antarctica PI12 |
title_full |
Biochemical Characterisation and Structure Determination of a Novel Cold-Active Proline Iminopeptidase from the Psychrophilic Yeast, Glaciozyma antarctica PI12 |
title_fullStr |
Biochemical Characterisation and Structure Determination of a Novel Cold-Active Proline Iminopeptidase from the Psychrophilic Yeast, Glaciozyma antarctica PI12 |
title_full_unstemmed |
Biochemical Characterisation and Structure Determination of a Novel Cold-Active Proline Iminopeptidase from the Psychrophilic Yeast, Glaciozyma antarctica PI12 |
title_sort |
biochemical characterisation and structure determination of a novel cold-active proline iminopeptidase from the psychrophilic yeast, glaciozyma antarctica pi12 |
publisher |
Multidisciplinary Digital Publishing Institute |
publishDate |
2022 |
url |
https://doi.org/10.3390/catal12070722 |
genre |
Antarc* Antarctica |
genre_facet |
Antarc* Antarctica |
op_source |
Catalysts; Volume 12; Issue 7; Pages: 722 |
op_relation |
Biocatalysis https://dx.doi.org/10.3390/catal12070722 |
op_rights |
https://creativecommons.org/licenses/by/4.0/ |
op_doi |
https://doi.org/10.3390/catal12070722 |
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Catalysts |
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